Kasetsart J. (Nat. Sci.) 42 : 118 - 126 (2008)

Influences of Anthraquinone Extraction Techniques from Morinda sp. on Extraction Efficiency

Wongwat Temiyaputra, Thidarat Suebsanga, Kesinee Yajom, Sirilak Piyaworanon and Noppol Leksawasdi*

ABSTRACT

The comparison of anthraquinone levels in root, stem, bark and leaf of Morinda sp. were investigated by HPLC with Alltima C8 column using 32%(v/v) acetonitrile and 0.5% (v/v) acetic acid as mobile phase at 40°C for 20 min. The roots contained the highest specific concentration of anthraquinone at 103.2 ± 4.8 mg/g dried mass, followed by stem (45.3 ± 6.3 mg/g), bark (6.90 ± 0.3 mg/ g) and leaf (1.20 ± 0.6 mg/g). Four extraction techniques were employed to extract anthraquinone from the roots of Morinda sp and the anthraquinone content was analyzed using spectrophotometer. Soxhlet apparatus was utilized in the first technique with 50% (v/v) methanol as an extraction solvent. It was found that 14.6 ± 1.0 mg/g dried root powder was extracted with the expenses of 3.97 ± 0.17 Baht/mg anthraquinone. This was compared to the second extraction method at room temperature with 80%(v/v) acetone and 50%(v/v) ethanol that led to the highest concentration of extracted anthraquinone at 38.9 ± 1.6 and 27.0 ± 6.9 mg/g with the accompanied cost of 2.94 ± 0.12 and 1.42 ± 0.36 Baht/mg, respectively. The third extraction procedure was carried out in the pressurised steamer at 1 bar and 100°C for 5 min with 80% (v/v) ethanol. The highest level of extracted anthraquinone was 95.3 ± 0.6 mg/g at the cost of 4.28 ± 0.03 Baht/mg. The last method of extraction was performed in the closed-circuit solid-liquid extraction unit at room temperature. Although the extraction cost was relatively small (1.50 ± 0.08 Baht/mg), the specific concentration obtained was not high (only 2.27 ± 0.11 mg/g). Key words: anthraquinone extraction, extraction cost, Morinda sp.

INTRODUCTION commonly found in plants such as Noni (Morinda sp.) and Cassod (Cassia sp.) (Kaewdok and Anthraquinone (Figure 1a) plays an Tubsombat, 2002). Noni plant or Indian Mulberry important role as a biocatalyst in the pulp paper tree is commonly available in the northern part of production industry. Ibrahim and Osman (1994) Thailand. discussed the potential of using anthraquinone as Shotipruk et al. (2004) examined the laxative and growth inhibitor of microbes such as anthraquinone extraction from M. citrifolia with fungi. Furthermore, anthraquinone can also be hot water at high pressure within the temperature used as substrate in the production of various dyes range of 110-220°C and flow rate speed of 2-6 and pigments such as alizarin. This chemical is ml/min. The best extraction condition was obtained

Department of Food Engineering, Faculty of Agro-Industry, Chiang Mai University, Chiang Mai, 50100, Thailand. * Corresponding author, e-mail: [email protected] Kasetsart J. (Nat. Sci.) 42(5) 119 at 220°C, 4 ml/min and 7 MPa with maximum industrial grade ethanol (OV chemical, Chiang anthraquinone level of 43 mg/g dried root. Mai, Thailand) were analytical reagent grade: Aobchey et al. (2002) reported anthraquinone absolute ethanol (Merck, Darmstadt, Germany, extraction from the root of M. augustifolia (Figure Product No. 1.00983.2500), acetone (Lab-Scan, 1b and 1c) and biomass obtained from root cell Dublin, Ireland, Cat. No. A3501), ethyl acetate culture. The extraction solvents were chloroform (Lab-Scan, Dublin, Ireland, Cat. No. A3511), and methanol at atmospheric pressure. The highest methanol (Merck, Darmstadt, Germany, Product level of anthraquinone obtained was 15 mg/g dried No. 106009) and anthraquinone (Fluka, New York, root which was compared to 8.9 mg/g of root cell USA, Cat. No. 2015490). The reasons of using culture. 95% industrial grade (86 Baht/kg) instead of an The aim of this research was to determine analytical grade absolute ethanol (452 Baht/kg) anthraquinone content in root, stem, bark and leaf were to lower the extraction cost and to provide of Morinda sp. in order to select the part of plant experimental data that reflected the practical large with the highest anthraquinone level for further scale extraction situation where industrial grade investigation on the extraction technique. The ethanol would be the more likely choice of effects of four extraction techniques, which extraction solvent. included (1) soxhlet extraction (2) simple solvent extraction at room temperature, (3) pressurized Sample preparation steamer extraction and (4) the extraction in closed- The roots of Morinda sp. with minimum circuit solid-liquid extraction unit, on the age of 3 yrs were collected from households within extraction cost and extraction efficiency were the Muang and Saraphee District of Chiang Mai examined so that the cost effective method of Province and Muang District of Lampoon anthraquinone extraction from Morinda sp. root Province during July - August. The samples were for large scale production could be established. kept frozen at -20°C before applying the appropriate size reduction methods for each part MATERIALS AND METHODS of plant. The harder portion, namely, root, stem and bark were chopped while the leaf was cut with Materials and chemicals knife into smaller pieces. This was followed by All of the chemicals used in the drying step in the tray dryer (Armfield, Ringwood, experiment with the exception of 95% (v/v) UK, Model UOP 8) at 65°C and air flow rate of 1

Figure 1 Structure of anthraquinone (a) (Kritsanapan and Nualkeaw, 2003) and roots of Morinda sp. before (b) and after (c) pretreatment for drying process. 120 Kasetsart J. (Nat. Sci.) 42(5) m/s until the equilibrium moisture content was was utilized in the comparison of anthraquinone reached. All of dried samples were fed into the content in root, stem, bark and leaf of Morinda hammer mill with a 250 µm sieve tray. sp.

Anthraquinone extraction with soxhlet Extraction in solid-liquid extraction unit apparatus The 5 g root powder was extracted with The original extraction method for C. 3 litres of distilled water in the closed circuit solid- siamea Britt. proposed by Kritsanapan and liquid extraction demonstration unit (Armfield, Nualkaew (2003) using soxhlet extraction unit Ringwood, UK, Model No. 12697) at room (Siripanya Trading, Lampang, Thailand) was temperature with pump speed of 13.5 l/h. Sample improved and modified for anthraquinone was collected every 30 min for 5 h. extraction from Morinda sp. root. Each extraction was repeated twice. The extraction was carried out Analytical methods with 150 ml of 50%(v/v) ethanol or 50%(v/v) The analysis of anthraquinone was methanol using four rounds of reflux followed by performed either spectrophotometrically by taking filtration with Whatman No. 4 filter paper. measurement from a spectrophotometer (Perkin Elmers, Waltham, USA, Model No. Lambda 25) Anthraquinone extraction with other methods at 325 nm for comparison of extraction efficiency Three replicates were used for each between each extraction method and extraction method by maintaining the ratio of root chromatographically using HPLC (Shimadzu, powder per volume of solvent at 0.17 g per 100 Japan) for the determination of anthraquinone ml. content in root, stem, bark and leaf of Morinda sp. with Alltima C8 column (Alltech, USA) at Extraction with different type of solvents at 40°C and a mobile phase flow rate of 2 ml/min room temperature whose composition was 32%(v/v) acetonitrile and Three types of solvents were used 0.5%(v/v) acetic acid. The injection volume and including ethanol, acetone and ethyl acetate. Each run time were 5 µl and 25 min, respectively. solvent was mixed with distilled water in the Anthraquinone peak appeared between 15.0 - 15.8 following %(v/v overall solution); 0, 5, 20, 35, min (Figure 2(a) and 2(b)) and was monitored at 50, 65, 80, 95 and 100. The extraction of 0.05 g 263 nm. The dilution with corresponding solvent Morinda sp. root powder with 30 ml of and appropriate dilution factor was applied each solvent was performed at room temperature accordingly. The quantification of anthraquinone for 6 h. in the sample was obtained from the standard curve of anthraquinone between 1.56 – 6.23 mg/ Extraction in pressurized steamer ml. Both methods of analyses were comparable The extraction was repeated as previous as evident from the relatively equivalent method with ethanol as a solvent. The sample was concentration of anthraquinone in root powder; placed in the moisture can and the extraction was 103.2 ± 4.8 mg/g for HPLC and 95.3 ± 0.6 mg/g carried out in the stainless steel pressurized for spectrophotometrical procedure. The steamer (All American, Miami, USA, Model determination of precision in terms of the No.1925x). The pressure and temperature were coefficient of variation (CV) and accuracy in terms controlled at 1 bar and 100°C respectively with of relative error for each method were extraction time of 5 min. The method of extraction subsequently determined as described by Skoog Kasetsart J. (Nat. Sci.) 42(5) 121

Figure 2 HPLC chromatogram of (a) anthraquinone standard and (b) sample from root powder. et al. (1996). The corresponding CV and relative RESULTS AND DISCUSSION error for the analysis of anthraquinone based on HPLC were found to be 6.59% for root powder Anthraquinone content in root, stem, bark and sample and -0.67% for anthraquinone standard, leaf of Morinda sp. by pressurized steam respectively. This was compared to the situation method of spectrophotometrical analysis where both The roots contained the highest specific parameters were 0.89% for CV and 0.29% for concentration of anthraquinone at 103.2 ± 4.8 mg/ relative error. The extraction cost was calculated g dried mass (Figure 3, (0.172 ± 0.008 mg/ml)/ from the initial cost involved in each extraction (0.05 g/30 ml) = 103.2 ± 4.8 mg/g), followed by method such as water, electricity, natural gas, stem (45.3 ± 6.3 mg/g), bark (6.90 ± 0.3 mg/g) materials and chemicals. The statistical hypothesis and leaf (1.20 ± 0.6 mg/g). This was compared to testing on difference in the level of extracted the maximum concentration of 1.2 mg/g anthraquinone and extraction cost were performed anthraquinone glycoside and 2.0 mg/g total using experimental mean comparison technique anthraquinone from C. siamea dried leaf (Skoog et al. 1996). (Kritsanapan and Nualkaew, 2003). 122 Kasetsart J. (Nat. Sci.) 42(5)

160

140

120

100

one (mg/g) one 80

60

40 Anthraquin 20

0 Root Stem Bark Leaf

Part of plant Figure 3 Anthraquinone content (HPLC method) in root, stem, bark and leaf of Morinda sp.

Extraction with soxhlet apparatus types of solvent. This might be described by a The improved soxhlet extraction method greater electrostatic force of water relative to other with 50%(v/v) ethanol and 50%(v/v) methanol molecules (CYBERLAB 2007, LESA 2007). yielded 11.2 ± 2.8 and 14.6 ± 1.0 mg anthraquinone Therefore, the extraction with solvents other than per g of dried root powder, respectively with 1 h water might also require a participating role of extraction time. The extraction expenses per 1 mg water at some extent to increase the extraction of extracted anthraquinone were 4.98 ± 1.25 and efficiency. Similar observation was also reported 3.97 ± 0.17 Baht, respectively. Hemwimol et al. by Sakunpak et al. (2007) who investigated the (2006) also performed a continuous extraction with roles of water in anthraquinone extraction from soxhlet method with only 200 ml absolute ethanol Senna alata. The concentration of water at 15% from 2 g of dried root powder for the period of 4 h (w/w) helped enhancing anthraquinone extraction at 70°C with extraction efficiency of 97.7%. by 1.47 times when compared to the situation Further comparative analysis with extraction in the where the water was absence. Hemwimol et al. pressurized steamer at 1 bar and 100°C for 5 min (2006) examined the optimal ratio of ethanol and with total anthraquinone level of 95.3 ± 0.6 mg/g water in anthraquinone extraction from Morinda dried root powder (Figure 6 at 80% ethanol, (0.159 sp. root by soaking 0.1 g of dried root powder in ± 0.001 mg/ml)/(0.05 g/30 ml) = 95.3 ± 0.6 mg/g) 10 ml of solvent. It was found that 50% ethanol indicated that soxhlet extraction methods had was able to extract anthraquinone at 1.88 times lower extraction efficiency than that of the greater than that of absolute ethanol. Such extraction in pressurized steamer. finding corresponded to the experimental results in Figure 4 (the highest level of 0.045 ± 0.009 mg/ Extraction with different type of solvents ml was obtained when 50% ethanol was used, The roles of water in the enhancement comparing to 0.037 ± 0.013 mg/ml for absolute of extraction efficiency were prominent in every ethanol) and Figure 5 (0.065 ± 0.003 mg/ml for case of anthraquinone extraction with various 80% acetone which was 2.6 times higher than Kasetsart J. (Nat. Sci.) 42(5) 123

Ethanol

0.20 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 Anthraquinone (mg/ml) 0.02 0.00 0 5 20 35 50 65 80 95 100

% Ethanol (v/v)

Figure 4 Anthraquinone extraction with a solvent consisted of ethanol and water at room temperature with various concentration level (%(v/v)). The analysis was carried out using spectrophotometer.

Acetone 0.20 0.18 0.16 0.14 0.12 0.10

inone (mg/ml) 0.08 0.06 0.04 Anthraqu 0.02 0.00 0 5 20 35 50 65 80 95 100

% Acetone (v/v)

Figure 5 Anthraquinone extraction with a solvent consisted of acetone and water at room temperature with various concentration level (%(v/v)). The analysis was carried out using spectrophotometer. 124 Kasetsart J. (Nat. Sci.) 42(5)

Extraction in pressurized steamer

0.20 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 Anthraquinone (mg/ml) 0.02 0.00 0 5 20 35 50 65 80 95 100 % Ethanol (v/v) [1 bar, 100oC]

Figure 6 Anthraquinone extraction in a pressurized steamer with ethanol and water at various concentration levels (%(v/v)) at 1 bar, 100°C for 5 min. The analysis was carried out using spectrophotometer.

0.025 ± 0.003 mg/ml for 100% acetone). Compared to other methods, the Hemwimol et al. (2006) explained that a relatively pressurized steamer extraction procedure yielded high polarity index (p.i.) of water (p.i. = 9) in the highest level of anthraquinone (0.159 ± 0.001 comparison to ethanol (p.i. = 5.2) might contribute mg/ml in Figure 6 which was equivalent to 95.3 ± to the enhancement of extraction efficiency. The 0.6 mg/g dried root powder). The temperature swelling of Morinda sp. root cells also increased increase accelerated diffusion process while the the surface area and facilitated the extraction rise in pressure encouraged the movement of efficiency by the solvents. In addition, the elevated molecules or ions in the extraction medium mass transfer coefficient due to the decreased (Jindawat, 2005) as evidence from the extraction ethanol viscosity in the presence of water for up of total soluble solid (TSS) from dried longan to 50% could all play parts in optimal extraction which found that boiling helped increase the at such situation. The significant role of acetone extraction speed and resulted in higher in boosting the effect of anthraquinone extraction concentration of extracted solute (Palakul and efficiency could be explained in term of higher Fongduang, 2006). The concentration of extracted mass transfer coefficient that allowed the better anthraquinone described in Figure 6, which was access of acetone to the anthraquinone reserve in later converted to mg/g, was higher than those of each cell of Morinda sp. Furthermore, the Aobchey et al. (2002) by 6.35 times (15 mg/g of similarity of carbonyl functional group (C=0) in dried root powder) and Shotipruk et al. (2004) by acetone to that of anthraquinone might also play 2.22 times (43 mg/g dried root powder). In part in the improvement of anthraquinone addition, Shotipruk et al. (2004) suggested that extraction efficiency (Hemwimol et al., 2006). the pressure level had no impact to the extraction Kasetsart J. (Nat. Sci.) 42(5) 125

Table 1 Summary of expenses involved in anthraquinone extraction with four extraction techniques. The anthraquinone content (mg/g) was determined by spectrophotometer. Extraction Solvent Extraction Anthraquinone Initial extraction method conditions (mg per g of cost of root powder) anthraquinone 1 mg (Baht) Soxhlet 50 %(v/v) ethanol Reflux 4 times 11.2 ± 2.8a 4.98 ± 1.25I Soxhlet 50 %(v/v) methanol Reflux 4 times 14.6 ± 1.0a 3.97 ± 0.17I Solvent 50 %(v/v) ethanol Room temperature 27.0 ± 6.9b 1.42 ± 0.36II Solvent 80 %(v/v) acetone Room temperature 38.9 ± 1.6c 2.94 ± 0.12III Solvent 50 %(v/v) EA* Room temperature 26.9 ± 5.1b 3.68 ± 0.70I,III PS# 80 %(v/v) ethanol 1 bar, 100°C 95.3 ± 0.6d 4.28 ± 0.03I SLE! Tap water Room temperature 0.0038 ± 0.0002e 1.50 ± 0.08II *ethyl acetate, #pressurized steamer, !solid-liquid extraction The number with the same alphabet (a-e) and Roman numerical (I – III) indicate no significant difference at 95% CI yield of anthraquinone within the temperature between extracted anthraquinone (mg/g root interval of 110-220°C. Such finding was in good powder) and the initial extraction cost of agreement with the current study where the high anthraquinone (Baht/mg) listed in Table 1. Even level of extracted anthraquinone was still obtained though, there was no statistical difference at 95% even at atmospheric pressure. Hemwimol et al. CI in the initial extraction cost between the soxhlet (2006) also illustrated that up to 96% of available and pressurized steamer, the cost ratio from both anthraquinone could be obtained when 80% methods differed by almost 10 times (2.25 for ethanol was used in the extraction system utilizing soxhlet method with 50% ethanol and 22.2 for ultrasound technology. However, it should be pressurized steamer method). noted that the error associated with this extraction procedure was still relatively large as evidence CONCLUSION from the presence of two outliers in Figure 6, in which the level of extracted anthraquinone (mg/ In conclusion, anthraquinone extraction ml) at 65 and 95% ethanol were much lower than in the pressurized steamer with 80% ethanol those at 50, 80 and 100% ethanol. Further provided the superior extraction in terms of experimentation should be conducted to extraction efficiency and cost ratio over that of a investigate whether extraction time beyond 5 min more expensive soxhlet and a less efficient solid- and extraction pressure (5 – 20 psi) play any part liquid extraction procedures. in stabilizing the extraction efficiency as well as the positioning or the number of extraction ACKNOWLEDGEMENTS containers presence in the pressurized steamer that might influence or interfere the accessing pattern The financial support of this research was of steam to each container. provided from “The Grant for New Generation Researcher” of Chiang Mai University (2005) to Comparison of anthraquinone extraction cost Noppol Leksawasdi and RPUS grant from Thai Further analysis of the best extraction Research Fund (TRF), Industrial Sector (2006) to method could be done by finding the cost ratio Wongwat Temiyaputra, Thidarat Suebsanga, 126 Kasetsart J. (Nat. Sci.) 42(5)

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